The phosphorylation of proteins plays a key role in the transduction of extracellular signals into the cell. The enzymes, called kinases, which effect such phosphorylations are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation. One of the major signal transduction pathways involves the enzyme protein kinase C (PKC), which is known to have a critical influence on cell proliferation and differentiation. PKC is activated by diacylglycerols (DAGs), which are metabolites released in signal transduction.
Interest in PKC was stimulated by the finding that PKC is the major, and perhaps only, cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells (Gescher et al., Anti-Cancer Drug Design 4:93-105 (1989)). Phorbols capable of tumor production can mimic the effect of DAG in activating PKC, suggesting that these tumor promoters act through PKC and that activation of this enzyme is at least partially responsible for the resulting tumorigenesis (Parker et al., Science 233:853-866 (1986)).
A number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents. PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs (Gescher, A. and Dale, I. L., Anti-Cancer Drug Design, 4:93-105 (1989)).
PKC is not a single enzyme, but a family of enzymes. At the present time at least seven isoforms (isozymes) of PKC have been identified: isoforms .alpha., .beta., and .gamma. have been purified to homogeneity, and isoforms .delta., .epsilon., .zeta. and .eta. have been identified by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishizuka, Nature, 334:661-665 (1988) for review) and may serve different physiological functions. For example, PKC-.gamma. seems to be expressed only in the central nervous system. PKC-.alpha. and -.beta. are expressed in most tissues, but have different patterns of expression in different cell types. For example, both PKC-.alpha. and PKC-.beta. are expressed in, and have been purified from, human epidermis. While PKC-.alpha. has been detected mainly in keratinocytes of the basal layers of the epidermis, PKC-.beta. is found mainly in the middle layers of the epidermis and Langerhans cells. PKC-.eta. has been found predominantly in the skin and lungs, with levels of expression much higher in these tissues than in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain (Osada et al., J Biol. Chem. 265:22434-22440 (1990)).
It is presently believed that different PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed.
For example, in psoriatic lesions there is an alteration in the ratio between PKC-.alpha. and PKC-.beta., with preferential loss of PKC-.beta. compared to normal skin (Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla., 1992).
One of the biological targets of PKC kinase activity is the c-jun protein encoded by the proto-oncogene c-jun. The c-jun protein is a major component of the AP-1 transcription factor (Bohmarm, D., et al., Science, 1987, 238, 1386-1392). Activation of protein kinase C results in dephosphorylation of the latent, phosphorylated form of c-jun protein, and subsequently, increased AP-1 activity (Boyle, W. J., et al., Cell, 1991, 8, 573-584).
Increased c-jun activity is associated with a wide variety of cancers and other diseases and conditions. Overexpression of c-jun has been associated with cancers including bladder carcinoma (Skopelitou, A., et al., Eur. Urol., 1997, 31, 464-471), lung carcinoma (Volm, M., et al., Clin. Exp. Metastasis, 1994, 12, 329-334), high-grade osteosarcomas (Franchi, A., et al., Virchows Arch., 1998, 432, 515-519), ovarian carcinoma (Spinner, D. M., et al., Int. J. Cancer, 1995, 63, 423-427), and tumors associated with the central nervous system, including medulloblastomas, neuroblastomas, astrocytomas and glioblastomas (Ferrer, I., et al., Neurosci. Lett., 1996, 214, 49-52). Overexpression of c-jun is also associated with Alzheimer's disease (Anderson, A. J., et al., J. Neurosci., 1996, 16, 1710-1719), while constitutive expression of c-jun is associated with rheumatoid arthritis (Herlitzka, D. S., et al., Ann. Rheum. Dis., 1996, 55, 298-304).
In the present invention, it has been determined that PKC-.alpha. is the isozyme associated with modulation of c-jun expression. It is believed that modulation of PKC-.alpha. expression will also aid in the treatment of the cancers and other diseases associated with c-jun.
Numerous compounds have been identified as PKC inhibitors (see Hidaka and Hagiwara, Trends in Pharm. Sci. 8:162-164 (1987) for review), though not all of these inhibit PKC specifically. The quinoline sulfonamide derivatives such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC at micromolar concentrations, though they exhibit similar enzyme inhibition kinetics for PKC and the CAMP-dependent and cGMP-dependent protein kinases. Staurosporine, an alkaloid product of Streptomyces sp., and its analogs, are the most potent in vitro inhibitors of PKC identified to date. However, they exhibit only limited selectivity among different protein kinases (Gescher, Anti-Cancer Drug Design 4:93-105 (1989)). Certain ceramides and sphingosine derivatives have been shown to have PKC inhibitory activity and to have promise for therapeutic uses, however, there remains a long-felt need for specific inhibitors of the enzymes.
Inhibitors of PKC may inhibit the activity or the synthesis (i.e. expression) of PKC-.alpha.. Specific inhibitors of the PKC-.alpha. isozyme include chemical compounds, monoclonal antibodies, protein fragments and antisense oligonucleotides. Go-6976 has been used as a specific PKC-.alpha. inhibitor to study the role of this enzyme in Alzheimer's disease (Benussi, L., et al., Neurosci. Lett., 1998, 240, 97-101), although others suggest that this compound is specific for both the .alpha. and .beta.1 isoforms (La Porta, Calif., et al., Br. J. Cancer, 1998, 78, 1283-1287). Isozyme-specific monoclonal antibodies have been used to study their role in various cellular processes (Liao, L. and Jaken., S., Cell Growth Differ., 1993, 4, 309-316; Chen, C. C., et al., Mol. Pharmacol., 1995, 48, 39-47). Parissenti, A. M., et al. (J. Biol. Chem., 1998, 273, 8940-8945) used PKC-.alpha. fusion proteins and deletion protein as specific PKC-.alpha. inhibitors. Godson et al. (J. Biol. Chem., 1993, 268, 11946-11950) disclosed use of stable transfection of antisense PKC-.alpha. cDNA in cytomegalovirus promotor-based expression vectors to specifically decrease expression of PKC-.alpha. protein by approximately 70%. It was demonstrated that this inhibition caused a loss of phospholipase A.sub.2 -mediated arachidonic acid release in response to the phorbol ester PMA. Attempts by the same researchers at inhibiting PKC activity with oligodeoxynucleotides were ultimately unsuccessful due to degradation of oligonucleotides. Ahmad et al. disclose that transfection of the human glioblastoma cell line, U-87, with vectors expressing antisense RNA to PKC.alpha. inhibits growth of the glioblastoma cells in vitro and in vivo. Ahmad et al., Neurosurg., 1994, 35, 904-908.
Fung, H., et al. (Cancer Res., 1997, 57, 3101-3105) purport to show that inhibition of PKC-.alpha. prevents asbestos-induced c-fos and c-jun expression. They demonstrate that PKC-.alpha. is the major isoform is a mesothelial cell line and that a general PKC inhibitor reduces c-fos and c-jun expression.
Thus, there is the need for improved methods and compositions for specific PKC-.alpha. inhibitors especially in modulation of c-jun expression.